S-Nitrosothiols (SNOs) signal a variety of biological activities in cells and tissues. Studies of SNO signaling are hampered by a lack of reliable assays. This has been particularly true for assays of SNO bonds and iron/porphyrin-containing cells and proteins, where autocapture of nitric oxide (NO) by iron after cleavage of the SNO bond alters/attenuates the assay signal. Though there is general agreement, for example, that a SNO bond is formed at the β93 cysteine of hemoglobin under physiological conditions, results vary substantially depending on assay technique. Moreover, efforts to protect against 1) artifactually measuring FeNO bonds (by photolysis); 2) artifactually forming FeNO or SNO bonds; and 3) artifactually dampening the signal by autocapture on the iron group, have led to complex assays requiring extensive hemoglobin pretreatment (with cyanide, N-ethyl-maleamide and a variety of other reagents) before the SNO bond can be assayed. These extreme chemical conditions have given rise to substantial controversy about issues such as whether SNO bond stability is affected by hemoglobin allostery. Moreover, the most reliable assays for SNO and FeNO bonds in hemoglobin and in other protein S-nitrosothiols require expensive and cumbersome equipment.
It has recently become evident that SNO signaling is deranged in a variety of human diseases, ranging from asthma to cystic fibrosis. SNO hemoglobin related signaling appears to be abnormal in sepsis, diabetes, pulmonary hypertension, and sickle cell anemia and congestive heart failure. Reliable SNO protein assays are likely to be relevant not only to in vitro cell culture work and physiology experiments, but also to clinical assays, particularly human clinical assays. Increased SNO loading and improved allosteric delivery of NO can improve perfusion to a variety of organs. On the other hand, when SNO loading is excessive, normal vasoregulation—particularly in the lung—can be blunted. Assays for NO hemoglobin may be of critical importance to improving health care over the next several decades, and it has been known for over a decade that NO circulates in the plasma of healthy humans.
A wide spectrum of analytical approaches and sample manipulation in recent years have yielded highly divergent values of S-nitrosothiol content in plasma or other fluids or tissues, often within a range of three orders of magnitude (see Giustarini et al., Clinica Chimica Acta, 2003, 330:85-98, for a review). For example, the various techniques and assays have found nitrosothiol levels ranging from 15 to 7,000 mmol/l, in serum or plasma.
S-nitrosothiols in biological samples have been measured by a wide spectrum of different techniques, e.g., chemiluminescence, calorimetric, amperometric, spectrophotometry, HPLC, GC-MS and fluorescence-based methods (Cook et al., Analytical Biochemistry, 1996, 238:150-158, Fang et al., Biochemical and Biophysical Research Communications, 1998, 252:535-540, Giustarini et al., Clinica Chimica Acta, 2003, 330:85-98, Jourd'Heuil et al., Free Radical Biology & Medicine, 2000, 28:3:409-417, Palmerini et al., Biology and Chemistry, 1998, 2:5:375-380, Palmerini et al., Biology and Chemistry, 1998, 2:5:375-380, Palmerini et al., Biology and Chemistry, 2000, 4:6:546-549, Park et al., Analytical Biochemistry, 1997, 249:61-66, Pfeiffer et al., Analytical Biochemistry, 1998, 258:68-73, Samouilov et al., Analytical Biochemistry, 1998, 258:322-330, Stubauer et al., Journal of Biological Chemistry, 1999, 274:40:28128-28133, and Tsikas et al., Journal of Chromatography, 2002, 772:335-346.). However, these techniques are not devoid of artifacts and the investigation of endogenous S-nitrosothiol levels is therefore hampered by methodological concerns.
For example, the spectrophotometric assay involves the colorimetric measurement of NO2− after displacement by HgCl2 by reaction with Griess solution. This technique is very simple, but has a relatively high detection limit (0.5 μmol/l). In addition, it has been suggested that mercury does not stoichiometrically release NO from RSNOs when added to plasma Assuming that plasma or tissue S-nitrosoprotein levels are very low, artifactual overestimation is a problem. Therefore, sample manipulation also assumes a critical role. The relative instability of RSNOs in some conditions has been demonstrated, in particular, in the presence of metals that can induce their decomposition. Moreover, it is known that the artifactual formation of S-nitrosothiols from nitrite and SH groups under acidic conditions occurs. In addition, the evaluation of RSNOs can be influenced by the occurrence of transnitrosation reactions interchanging the NO+ moiety between two sulfhydryl groups. S-nitrosothiol assays must also take into account the high background concentration of nitrites present in biological samples, which can be responsible for some overestimated determinations. This anion can be removed by reaction with ammonium sulfamate via their reduction to nitrogen gas.
There is a long felt need in the art for a reliable and accurate method of detecting or measuring S-nitrosothiols in the broad range of samples researchers and medical practitioners encounter without need to change the method for different types of samples. The present invention satisfies this need.